Composition of proteoglycans synthesized by rabbit aortic explants in culture and the effect of experimental atherosclerosis

The synthesis of proteoglycans by aorta explants from rabbits with diet-induced atherosclerosis and controls was studied by 35S-incorporation. Proteoglycans were isolated under dissociative conditions from incubation medium and from arterial explants. Additionally, the tissue proteoglycans that were not extracted by 4 M guanidine-HCl were solubilized by digestion of the tissue by elastase in the presence of proteinase inhibitors. The residual tissue was hydrolyzed by papain and glycosaminoglycans were isolated. The atherosclerotic aorta tissue incorporated twice the amount of 35S into proteoglycans than observed for controls; in both groups about 70% of the label incorporated into the tissue was noted in the proteoglycans extracted by guanidine-HC;, while about 30% of the total 35S-labeled proteoglycans synthesized by the explants were found in the media. Atherosclerotic tissue incorporated 35S predominantly into chondroitin sulfate proteoglycans when compared to control tissue. The chondroitinase ABC-digestable proteoglycans that were extracted by guanidine-HCl from atherosclerotic tissues were of larger molecular size than those from control tissue, but the core proteins from these preparations were similar. The heparan sulfate proteoglycan that was obtained by dissociative extraction from atherosclerotic tissue had greater amounts of N-acetyl and lesser amounts of N-sulfate ester groups than the preparation from control tissue. Digestion of the tissue by elastase yielded heparan sulfate proteoglycan as the major constituent in both groups, although atherosclerotic tissue contained relatively small amounts of this proteoglycan. The residual tissue from both groups contained chondroitin sulfate and heparan sulfate as the major glycosaminoglycans with the latter showing a decrease with atherosclerosis. Atherosclerotic tissue secreted into the medium about two-fold more 35S-labeled proteoglycans with larger molecular size than control tissue; proteoglycans of the heparan sulfate and chondroitin sulfate types were the major constituents in the culture medium of both tissues. Thus, proteoglycans undergo both quantitative and qualitative changes in atherosclerosis, reflecting the enhanced smooth muscle cell activity. These changes are potentially important in modulating lipoprotein binding and hemostatic properties, as well as fibrillogenesis of the arterial wall.     

An Improved Preparation of Rp and Sp N6, N6, O2′ - Tri-Benzoyl-Adenosine-3′, 5′-Cyclic Phosphoranilidates

Abstract

The conversion of the benzoylated cAMP (2) to the diastereomeric mixture of the anilidates (3) was improved. Replacing the triphenylphosphine/carbon tetrachloride mixture by oxalyl chloride in the presence of a catalytic amount of DMF followed by the addition of aniline not only increased the yield from 27 to 96% but rendered much easier the separation of the diastereoisomers (3), which were formed in approximately equal amounts. This greatly improved the accessability of Rp and Sp-cAMPS (1).     

Food provision to nestlings in the Hoopoe Upupa epops: implications for the conservation of a small endangered population in the Swiss Alps

In an attempt to recognize the possible ecological causes of the decline of a population of Hoopoes Upupa epops in the Swiss Alps, we collected data on resource exploitation. The prey provisioned to nestlings by parents was investigated at four breeding sites using photographs (n = 4353, 80% of which enabled prey identification). Molecrickets Gryllotalpa gryllotalpa and Lepidoptera (larvae and pupae) were dominant in nestling diet (93% frequency; 97% biomass). Although Molecrickets were provisioned less frequently (26%) than Lepidoptera (67%), they represented 68% of the total biomass (vs 29% for Lepidoptera). There was an overall negative relationship between the proportion of Molecricket biomass in the diet and the parents' feeding rate, whereas a comparison between broods showed that a higher provisioning activity did not lead to an increase in the biomass supplied to the chicks. A diet based on Molecrickets therefore appears to be energetically advantageous. As Molecrickets are a traditional prey of Hoopoes in central Europe, this might be relevant to other populations. In the study area, Molecrickets occur only on the intensively cultivated plain, whereas the majority of Hoopoe pairs nest at various altitudes on the foothills adjacent to the plain as the latter provides at present almost no suitable nesting sites. Hoopoes breeding higher up on the foothills seem to experience greater provisioning costs and have, on average, lower breeding success. Providing nest sites on the plain is the main conservation measure proposed for the local Hoopoe population. Further attention should also be paid to Molecrickets as these may be crucial for Hoopoes.     

Wide distribution of the cysteine string proteins in Drosophila tissues revealed by targeted mutagenesis

The “cysteine string protein” (CSP) genes of higher eukaryotes code for a novel family of proteins characterized by a “J” domain and an unusual cysteine-rich region. Previous studies had localized the proteins in neuropil and synaptic terminals of larval and adult Drosophila and linked the temperature-sensitive paralysis of the mutants described here to conditional failure of synaptic transmission. We now use the null mutants as negative controls in order to reliably detect even low concentrations of CSPs by immunohistochemistry, employing three monoclonal antibodies. In wild-type flies high levels of cysteine string proteins are found not only in apparently all synaptic terminals of the embryonic, larval, and adult nervous systems, but also in the “tall cells” of the cardia, in the follicle cells of the ovary, in specific structures of the female spermatheca, and in the male testis and ejaculatory bulb. In addition, low levels of CSPs appear to be present in all tissues examined, including neuronal perikarya, axons, muscles, Malpighian tubules, and salivary glands. Western blots of isolated tissues demonstrate that of the four isoforms expressed in heads only the largest is found in non-neural organs. The wide expression of CSPs suggests that at least some of the various phenotypes of the null mutants observed at permissive temperatures, such as delayed development, short adult lifespan, modified electroretinogram, and optomotor behavior, may be caused by the lack of CSPs outside synaptic terminals.     

Cyclic Expression of A Nuclear Protein In A Dinoflagellate

Nuclei of the dinoflagellate Crypthecodinium cohnii strain Whd were isolated and nuclear proteins were extracted in three fractions, corresponding to the increasing affinity of these proteins to genomic DNA. One fraction contained two major bands (48- and 46-kDa) and antibodies specific to this fraction revealed two major bands by Western blot on nuclear extracts, corresponding to the 46- and 48-kDa bands. The 48-kDa protein was detected in G1 phase but not in M phase cells. An expression cDNA library of C. cohnii was screened with these antibodies, and two different open reading frames were isolated. Dinoflagellate nuclear associated protein (Dinap1), one of these coding sequences, was produced in E. coli and appeared to correspond to the 48-kDa nuclear protein. No homologue of this sequence was found in the data bases, but two regions were identified, one including two putative zinc finger repeats, and one coding for two potential W/W domains. The second coding sequence showed a low similarity to non-specific sterol carrier proteins. Immunocytolocalization with specific polyclonal antibodies to recombinant Dinap1 showed that the nucleus was immunoreactive only during the G1 phase: the nucleoplasm was immunostained, while chromosome cores and nuclear envelopes were negative.     

A historical biogeography of megadiverse Sericini—another story “out of Africa”?

Megadiverse insect groups present special difficulties for biogeographers because poor classification, incomplete knowledge of taxonomy, and many undescribed species can introduce a priori sampling bias to any analysis. The historical biogeography of Sericini, a tribe of melolonthine scarabs comprising about 4000 species, was investigated using the most comprehensive and time‐calibrated molecular phylogeny available today. Problems arising through nomenclatural confusion were overcome by extensive sampling (665 species) from all major lineages of the tribe. A West Gondwanan origin of Sericini (c. 112 Ma) was reconstructed using maximum parsimony, maximum‐likelihood and model‐based ancestral area estimation. Vicariance in the tribe's earliest history separated Neotropical and Old World Sericini, whereas subsequent lower Cretaceous biogeography of the tribe was characterized by repeated migrations out of Africa, resulting in the colonization of Eurasia and Madagascar. North America was colonized from Asia during the Cenozoic and a lineage of “Modern Sericini” reinvaded Africa. Diversification dynamics revealed three independent shifts to increased speciation rates: in African ant‐adapted Trochalus, Oriental Tetraserica, and Asian and African Sericina. Southern Africa is proposed as both cradle and refuge of Sericini. This area has retained many old lineages that portray the evolution of the African Sericini fauna as a series of taxon pulses.     

Weak functional coupling of the melanocortin-1 receptor expressed in human adipocytes

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40-49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-alpha), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-alpha upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R-effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.     

Large-scale identification of polymorphic microsatellites using an <Emphasis Type="Italic">in silico</Emphasis> approach

Background  

Simple Sequence Repeat (SSR) or microsatellite markers are valuable for genetic research. Experimental methods to develop SSR markers are laborious, time consuming and expensive. In silico approaches have become a practicable and relatively inexpensive alternative during the last decade, although testing putative SSR markers still is time consuming and expensive. In many species only a relatively small percentage of SSR markers turn out to be polymorphic. This is particularly true for markers derived from expressed sequence tags (ESTs). In EST databases a large redundancy of sequences is present, which may contain information on length-polymorphisms in the SSR they contain, and whether they have been derived from heterozygotes or from different genotypes. Up to now, although a number of programs have been developed to identify SSRs in EST sequences, no software can detect putatively polymorphic SSRs.